Method for making a high oxygen affinity modified hemoglobin for oxygen transport

ABSTRACT

The present invention relates to blood products, and more particularly to compositions comprising a modified oxygenated hemoglobin having a high affinity for oxygen and methods for making such compositions. Such compositions according to the present invention have better stability to autooxidation and superior oxygen carrying characteristics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. Ser. No.11/088,934, filed Mar. 23, 2005, which is a continuation of U.S. Ser.No. 10/925,067, filed Aug. 24, 2004 now U.S. Pat. No. 6,974,795, whichis a continuation of U.S. Ser. No. 10/340,141, filed Jan. 10, 2003 nowU.S. Pat. No. 6,844,317, which is a continuation-in-part of U.S. Ser.No. 10/114,400, filed Apr. 1, 2002, now abandoned, and claims priorityunder 35 U.S.C. §119(e) to U.S. Ser. No. 60/347,741, filed Jan. 11,2002.

TECHNICAL FIELD

The present invention relates to blood products, and more particularlyto compositions comprising a modified hemoglobin having a high affinityfor oxygen and methods for making such compositions.

BACKGROUND OF THE INVENTION

The Circulatory System and the Nature of Hemoglobin

The blood is the means for delivering nutrients to the tissues andremoving waste products from the tissues for excretion. The blood iscomposed of plasma in which red blood cells (RBCs or erythrocytes),white blood cells (WBCs), and platelets are suspended. Red blood cellscomprise approximately 99% of the cells in blood, and their principalfunction is the transport of oxygen to the tissues and the removal ofcarbon dioxide therefrom.

The left ventricle of the heart pumps the blood through the arteries andthe smaller arterioles of the circulatory system. The blood then entersthe capillaries, where the majority of the exchange of nutrients andcellular waste products occurs. (See, e.g., A. C. Guyton, “HumanPhysiology And Mechanisms Of Disease” (3rd. ed.; W. B. Saunders Co.,Philadelphia, Pa.), pp. 228-229 (1982)). Thereafter, the blood travelsthrough the venules and veins in its return to the right atrium of theheart. Though the blood that returns to the heart is oxygen-poorcompared to that which is pumped from the heart, when at rest, thereturning blood still contains about 75% of the original oxygen content.

The reversible oxygenation function (i.e., the delivery of oxygen) ofRBCs is carried out by the protein hemoglobin. In mammals, hemoglobinhas a molecular weight of approximately 64,000 daltons and is composedof about 6% heme and 94% globin. In its native form, it contains twopairs of subunits (i.e., it is a tetramer), each containing a heme groupand a globin polypeptide chain. In aqueous solution, hemoglobin ispresent in equilibrium between the tetrameric (MW 64,000) and dimericforms (MW 32,000); outside of the RBC, the dimers are prematurelyexcreted by the kidney (plasma half-life of approximately 2-4 hours).Along with hemoglobin, RBCs contain stroma (the RBC membrane), whichcomprises proteins, cholesterol, and phospholipids.

Exogenous Blood Products

Due to the demand for blood products in hospitals and other settings,extensive research has been directed at the development of bloodsubstitutes and plasma expanders. A blood substitute is a blood productthat is capable of carrying and supplying oxygen to the tissues. Bloodsubstitutes have a number of uses, including replacing blood lost duringsurgical procedures and following acute hemorrhage, and forresuscitation procedures following traumatic injury. Plasma expandersare blood substitutes that are administered into the vascular system butare typically not capable of carrying oxygen. Plasma expanders can beused, for example, for replacing plasma lost from burns, to treat volumedeficiency shock, and to effect hemodilution (e.g., for the maintenanceof normovolemia and to lower blood viscosity). Essentially, bloodsubstitutes can be used for these purposes or any purpose in whichbanked blood is currently administered to patients. (See, e.g., U.S.Pat. Nos. 4,001,401 to Bonson et al., and U.S. Pat. No. 4,061,736 toMorris et al.)

The current human blood supply is associated with several limitationsthat can be alleviated through the use of an exogenous blood substitute.To illustrate, the widespread availability of safe and effective bloodsubstitutes would reduce the need for banked (allogeneic) blood.Moreover, such blood substitutes would allow the immediate infusion of aresuscitation solution following traumatic injury without regard tocross-matching (as is required for blood), thereby saving valuable timein resupplying oxygen to ischemic tissue. Likewise, blood substitutescan be administered to patients prior to surgery, allowing removal ofautologous blood from the patients which could be returned later in theprocedure, if needed, or after surgery. Thus, the use of exogenous bloodproducts not only protects patients from exposure to non-autologous(allogeneic) blood, it conserves either autologous or allogeneic(banked, crossmatched) blood for its optimal use.

Limitations of Current Blood Substitutes

Attempts to produce blood substitutes (sometimes referred to as“oxygen-carrying plasma expanders”) have thus far produced products withmarginal efficacy or whose manufacture is tedious, expensive, or both.Frequently, the cost of manufacturing such products is so high that iteffectively precludes the widespread use of the products, particularlyin those markets where the greatest need exists (e.g., emergingthird-world economies).

Blood substitutes can be grouped into the following three categories: i)perfluorocarbon-based emulsions, ii) liposome-encapsulated hemoglobin,and iii) modified cell-free hemoglobin. As discussed below, none hasbeen entirely successful, though products comprising modified cell-freehemoglobin are thought to be the most promising. Perfluorochemical-basedcompositions dissolve oxygen as opposed to binding it as a ligand. Inorder to be used in biological systems, the perfluorochemical must beemulsified with a lipid, typically egg-yolk phospholipid. Though theperfluorocarbon emulsions are inexpensive to manufacture, they do notcarry sufficient oxygen at clinically tolerated doses to be effective.Conversely, while liposome-encapsulated hemoglobin has been shown to beeffective, it is far too costly for widespread use. (see generally,Winslow, Robert M., “Hemoglobin-based Red Cell Substitutes,” JohnsHopkins University Press, Baltimore (1992)).

Most of the blood substitute products in clinical trials today are basedon modified hemoglobin. These products, frequently referred to ashemoglobin-based oxygen carriers (HBOCs), generally comprise ahomogeneous aqueous solution of a chemically-modified hemoglobin,essentially free from other red cell residue (stroma). Althoughstroma-free hemoglobin (SFH) from humans is the most common raw materialfor preparing a HBOC, other sources of hemoglobin have also been used.For example, hemoglobin can be obtained or derived from animal blood(e.g., bovine or porcine hemoglobin) or from bacteria or yeast ortransgenic animals or plants molecularly altered to produce a desiredhemoglobin product.

The chemical modification is generally one of intramolecularcross-linking, oligomerization and/or polymer conjugation to modify thehemoglobin such that its persistence in the circulation is prolongedrelative to that of unmodified hemoglobin, and its oxygen bindingproperties are similar to those of blood. Intramolecular cross-linkingchemically binds together subunits of the tetrameric hemoglobin unit toprevent the formation of dimers which, as previously indicated, areprematurely excreted. (See, e.g., U.S. Pat. No. 5,296,465 to Rausch etal.)

The high costs of manufacturing HBOC products have greatly limited theircommercial viability. In addition, the present inventors have found thatknown HBOCs have a tendency to release excessive amounts of oxygen tothe tissues at the arteriole walls rather than the capillaries. This canresult in insufficient oxygen available for delivery by the HBOC to thetissues surrounding the capillaries. This is despite the fact that theinitial loading of the HBOC with oxygen may be relatively high, comparedwith that normally achieved with natural red blood cells, except in thecase of very low affinity mutants.

In most instances, HBOCs have been designed to have oxygen affinitiesthat are the same as or lower than that of native hemoglobin. However,as discussed above, this may result in insufficient delivery of oxygento the tissues. Accordingly, the present invention relates to a bloodsubstitute that comprises an HBOC with high oxygen affinities in anaqueous diluent.

SUMMARY OF THE INVENTION

The methods and compositions of the present invention are useful in avariety of settings including emergency rooms, operating rooms, militaryconflicts, cancer hospitals, and veterinary clinics. The low toxicityand high stability of the present invention permits storage at roomtemperature without compromising the efficacy of the described bloodsubstitute. The present invention also circumvents the requirement forblood-type cross-matching and the associated laboratory testing,allowing for earlier and safer intervention in patient treatment. Thecombination of low toxicity, long-term stability, and universalapplicability of the present invention therefore presents a particularlyuseful substitute for blood.

In one aspect, the present invention provides a blood substitute productcomprising surface-modified oxygenated hemoglobin, wherein thesurface-modified oxygenated hemoglobin has a P50 less than nativestroma-free hemoglobin from the same animal source (i.e. from the samespecies of animal) when measured under the same conditions. Suitableanimal sources include, e.g., humans, cows, pigs, horses.

In a preferred embodiment, the blood substitute product takes the formof a composition comprising the surface-modified oxygenated hemoglobinand an aqueous diluent.

In a specific embodiment, the surface-modified oxygenated hemoglobin hasa P50 less than 10 torr, preferably less than 7 torr.

In another aspect, the present invention provides a blood substituteproduct produced by covalently attaching one or more polyalkylene oxidesto the oxygenated hemoglobin.

In a specific embodiment, the blood substitute product is produced bycovalently attaching a polymer of a polyalkylene oxide such aspolyethylene glycol (PEG) having the formula H(OCH₂CH₂)_(n)OH, where nis greater than or equal to 4. Preferably, the product has amethemoglobin/total hemoglobin ratio less than 0.10.

In yet another aspect, the present invention provides a blood substituteproduct stable to autooxidation at 24° C., comprising a PEG-hemoglobinconjugate, wherein the methemoglobin/total hemoglobin ratio is less than0.10 and the PEG-hemoglobin conjugate has a P50 less than 10 torr.

In another aspect, the present invention provides a method of making ablood substitute composition comprising the steps of: a) preparinghemoglobin having a methemoglobin/total hemoglobin ratio less than 0.10;b) covalently attaching polyalkylene oxide to the hemoglobin to formsurface-modified oxygenated hemoglobin having a P50 less than 10 torr;and c) suspending the surface-modified oxygenated hemoglobin in asuitable diluent. Preferably preparing hemoglobin further comprisesisolating hemoglobin from red blood cells.

In addition, the step of preparing the hemoglobin can further compriseisolating hemoglobin from red blood cells, wherein the hemoglobin has amethemoglobin/total hemoglobin ratio of 0.10 or greater, and exposingthe hemoglobin to aerobic conditions (i.e. to the atmosphere) for a timesufficient to lower the methemoglobin/total hemoglobin ratio to lessthan 0.10. This step may be carried out in the absence of athiol-containing reducing agent.

In yet another aspect, the present invention provides a method of usinga blood substitute product to deliver oxygen to a tissue, comprisingadministering the product in an aqueous diluent to a mammal in needthereof.

Other aspects of the present invention are described throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the FPLC chromatogram of MalPEG-Hb and SFH.

FIG. 2 depicts oxygen equilibrium curves for MalPEG-Hb and SFH.

FIG. 3 depicts FPLC patterns of elution for the two PEG-modifiedhemoglobins (PHP and POE) and unmodified hemoglobin (SFH). Note that thepatterns for PHP and POE are qualitatively, but not quantitatively,similar. Also, note the small peak of apparently unmodified hemoglobinin the POE curve.

FIG. 4 depicts oxygen equilibrium curves for the two PEG-modifiedhemoglobins (PHP and POE). Note that neither has significantcooperativity.

FIG. 5 depicts the rate of oxidation over time when MalPEG-hemoglobin isat room temperature. Samples were measured in duplicate from 2 separatebottles stored in the same way. The rate of oxidation is 1 percent perhour of total hemoglobin, going from 5.0 to 5.5 percent in 10 hours.

FIG. 6 depicts Kaplan-Meier survival analysis of the two groups ofanimals that received either PHP or POE.

FIG. 7 depicts mean arterial pressure in animals that received the twoPEG-modified hemoglobins (PHP and POE). The response is immediate andgreater in the animals that received PHP. However, pressure is bettermaintained in the POE animals during the hemorrhage period.

FIG. 8 depicts a summary of various rates of oxidation over time whenMalPEG-Hb is stored for six days at −20° C., five days at +4° C., andten hours at room temperature (24° C.).

FIG. 9 depicts the rate of oxidation over time when MalPEG-Hb is storedfor five days at +4° C.

FIG. 10 depicts the rate of oxidation over time when MalPEG-Hb is storedfor ten hours at room temperature.

DESCRIPTION OF THE INVENTION

The present invention is directed to blood substitutes comprising HBOCshaving high oxygen affinity. For certain applications, thesecompositions are capable of delivering oxygen to tissues moreefficiently than blood substitutes with oxygen affinities thatapproximate native hemoglobin.

Definitions

To facilitate understanding of the invention set forth in the disclosurethat follows, a number of terms are defined below.

The term “hemoglobin” refers generally to the protein contained withinred blood cells that transports oxygen. Each molecule of hemoglobin has4 subunits, 2α chains and 2 β chains, which are arranged in a tetramericstructure. Each subunit also contains one heme group, which is theiron-containing center that binds oxygen. Thus, each hemoglobin moleculecan bind 4 oxygen molecules.

The term “modified hemoglobin” includes, but is not limited to,hemoglobin altered by a chemical reaction such as intra- andinter-molecular cross-linking, genetic manipulation, polymerization,and/or conjugation to other chemical groups (e.g., polyalkylene oxides,for example polyethylene glycol, or other adducts such as proteins,peptides, carbohydrates, synthetic polymers and the like). In essence,hemoglobin is “modified” if any of its structural or functionalproperties have been altered from its native state. As used herein, theterm “hemoglobin” by itself refers both to native, unmodified,hemoglobin, as well as modified hemoglobin.

The term “surface-modified hemoglobin” is used to refer to hemoglobindescribed above to which chemical groups such as dextran or polyalkyleneoxide have been attached, most usually covalently. The term “surfacemodified oxygenated hemoglobin” refers to hemoglobin that is in the “R”state when it is surface modified.

The term “stroma-free hemoglobin” refers to hemoglobin from which allred blood cell membranes have been removed.

The term “methemoglobin” refers to an oxidized form of hemoglobin thatcontains iron in the ferric state and cannot function as an oxygencarrier.

The term “MalPEG-Hb” refers to hemoglobin to which malemidyl-activatedPEG has been conjugated. Such MalPEG may be further referred to by thefollowing formula:Hb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—O—CH₃)_(m)  Formula I

where Hb refers to tetrameric hemoglobin, S is a surface thiol group, Yis the succinimido covalent link between Hb and Mal-PEG, R is an alkyl,amide, carbamate or phenyl group (depending on the source of rawmaterial and the method of chemical synthesis), [O—CH₂—CH₂]_(n) are theoxyethylene units making up the backbone of the PEG polymer, where ndefines the length of the polymer (e.g., MW=5000), and O—CH₃ is theterminal methoxy group. PHP and POE are two different PEG-modifiedhemoglobin.

The term “perfluorocarbons” refers to synthetic, inert, molecules thatcontain fluorine atoms, and that consist entirely of halogen (Br, F, Cl)and carbon atoms. In the form of emulsions, they are under developmentas blood substances, because they have the ability to dissolve manytimes more oxygen than equivalent amounts of plasma or water.

The term “plasma expander” refers to any solution that may be given to asubject to treat blood loss.

The term “oxygen carrying capacity,” or simply “oxygen capacity” refersto the capacity of a blood substitute to carry oxygen, but does notnecessarily correlate with the efficiency in which it delivers oxygen.Oxygen carrying capacity is generally calculated from hemoglobinconcentration, since it is known that each gram of hemoglobin binds 1.34ml of oxygen. Thus, the hemoglobin concentration in g/dl multiplied bythe factor 1.34 yields the oxygen capacity in ml/dl. Hemoglobinconcentration can be measured by any known method, such as by using theβ-Hemoglobin Photometer (HemoCue, Inc., Angelholm, Sweden). Similarly,oxygen capacity can be measured by the amount of oxygen released from asample of hemoglobin or blood by using, for example, a fuel cellinstrument (e.g., Lex-O₂-Con; Lexington Instruments).

The term “oxygen affinity” refers to the avidity with which an oxygencarrier such as hemoglobin binds molecular oxygen. This characteristicis defined by the oxygen equilibrium curve which relates the degree ofsaturation of hemoglobin molecules with oxygen (Y axis) with the partialpressure of oxygen (X axis). The position of this curve is denoted bythe value, P50, the partial pressure of oxygen at which the oxygencarrier is half-saturated with oxygen, and is inversely related tooxygen affinity. Hence the lower the P50, the higher the oxygenaffinity. The oxygen affinity of whole blood (and components of wholeblood such as red blood cells and hemoglobin) can be measured by avariety of methods known in the art. (See, e.g., Winslow et al., J.Biol. Chem. 252(7):2331-37 (1977)). Oxygen affinity may also bedetermined using a commercially available HEMOX™ TM Analyzer (TCSScientific Corporation, New Hope, Pa.). (See, e.g., Vandegriff andShrager in “Methods in Enzymology” (Everse et al., eds.) 232:460(1994)).

The terms “hypertonic” means a colloidal solution with a colloidalosmotic pressure (oncotic) than blood (>approximately 25-27 mm Hg).Colloid osmotic pressure may be measured by any suitable technique, suchas in a Wescor instrument.

The term “oxygen-carrying component” refers broadly to a substancecapable of carrying oxygen in the body's circulatory system anddelivering at least a portion of that oxygen to the tissues. Inpreferred embodiments, the oxygen-carrying component is native ormodified hemoglobin, and is also referred to herein as a “hemoglobinbased oxygen carrier,” or “HBOC”.

The term “hemodynamic parameters” refers broadly to measurementsindicative of blood pressure, flow and volume status, includingmeasurements such as blood pressure, cardiac output, right atrialpressure, and left ventricular end diastolic pressure.

The term “crystalloid” refers to small molecules (usually less than 10Å) such as salts, sugars, and buffers. Unlike colloids, crystalloids donot contain any oncotically active components and equilibrate in betweenthe circulation and interstitial spaces very quickly.

The term “colloid,” in contrast to “crystalloid” refers to largermolecules (usually greater than 10 Å) that equilabrate across biologicalmembranes depending on their size and charge and includes proteins suchas albumin and gelatin, as well as starches such as pentastarch andhetastarch.

The term “colloid osmotic pressure” refers to the pressure exerted by acolloid to equilibrate fluid balance across a membrane.

The term “stable to autooxidation” refers to the ability of a HBOC tomaintain a low rate of autoxidation. HBOC is considered stable at 24° C.if the methemoglobin/total hemoglobin ratio does not increase more than2% after 10 hours at 24° C. For example, if the rate of autoxidation is0.2 hr⁻¹, then if the initial percentage of methemoglobin is 5%, HBOCwould be considered stable at room temperature for 10 hours if thispercentage did not increase above 7%.

The term “methemoglobin/total hemoglobin ratio” refers to the ratio ofdeoxygenated hemoglobin to total hemoglobin.

The term “mixture” refers to a mingling together of two or moresubstances without the occurrence of a reaction by which they would losetheir individual properties; the term “solution” refers to a liquidmixture; the term “aqueous solution” refers to a solution that containssome water and may also contain one or more other liquid substances withwater to form a multi-component solution; the term “approximately”refers to the actual value being within a range, e.g. 10%, of theindicated value.

The term “polyethylene glycol” refers to liquid or solid polymers of thegeneral chemical formula H(OCH₂CH₂)_(n)OH, where n is greater than orequal to 4. Any PEG formulation, substituted or unsubstituted, can beused.

The meaning of other terminology used herein should be easily understoodby someone of reasonable skill in the art.

The Nature of Oxygen Delivery and Consumption

Although the successful use of the compositions and methods of thepresent invention do not require comprehension of the underlyingmechanisms of oxygen delivery and consumption, basic knowledge regardingsome of these putative mechanisms may assist in understanding thediscussion that follows. It has generally been assumed that thecapillaries are the primary conveyors of oxygen to the tissue. However,regarding tissue at rest, current findings indicate that there isapproximately an equipartition between arteriolar and capillary oxygenrelease. That is, hemoglobin in the arterial system is believed todeliver approximately one third of its oxygen content in the arteriolarnetwork and one-third in the capillaries, while the remainder exits themicrocirculation via the venous system.

The arteries themselves are sites of oxygen utilization. For example,the artery wall requires energy to effect regulation of blood flowthrough contraction against vascular resistance. Thus, the arterial wallis normally a significant site for the diffusion of oxygen out of theblood. However, current oxygen-delivering compositions (e.g., HBOCs) mayrelease too much of their oxygen content in the arterial system, andthereby induce an autoregulatory reduction in capillary perfusion.Accordingly, the efficiency of oxygen delivery of a blood substitute mayactually be hampered by having too much oxygen or too low an oxygenaffinity.

The rate of oxygen consumption by the vascular wall, i.e., thecombination of oxygen required for mechanical work and oxygen requiredfor biochemical synthesis, can be determined by measuring the gradientat the vessel wall. See, e.g., Winslow, et al., in “Advances in BloodSubstitutes” (1997), Birkhauser, ed., Boston, Mass., pages 167-188.Present technology allows accurate oxygen partial pressure measurementsin a variety of vessels. The measured gradient is directly proportionalto the rate of oxygen utilization by the tissue in the region of themeasurement. Such measurements show that the vessel wall has a baselineoxygen utilization which increases with increases in inflammation andconstriction, and is lowered by relaxation.

The vessel wall gradient is inversely proportional to tissueoxygenation. Vasoconstriction increases the oxygen gradient (tissuemetabolism), while vasodilation lowers the gradient. Higher gradientsare indicative of the fact that more oxygen is used by the vessel wall,while less oxygen is available for the tissue. The same phenomenon isbelieved to be present throughout the microcirculation.

The Relationship between Vasoconstriction and Oxygen Affinity

The rationale for developing an HBOC with high oxygen affinity is based,in part, on past studies using cell-free hemoglobins as alternatives tored blood cell transfusions. Some of the physiological effects of thesesolutions remain incompletely understood. Of these, perhaps the mostcontroversial is the propensity to cause vasoconstriction, which may bemanifest as hypertension in animals and man (Amberson, W., “Clinicalexperience with hemoglobin-saline solutions,”. Science 106: 117-117(1947)) (Keipert, P., A. Gonzales, C. Gomez, V. Macdonald, J. Hess, andR. Winslow, “Acute changes in systemic blood pressure and urine outputof conscious rats following exchange transfusion withdiaspirin-crosslinked hemoglobin solution,” Transfusion 33: 701-708,(1993)). Human hemoglobin crosslinked between α chains withbis-dibromosalicyl-fumarate (ααHb) was developed by the U.S. Army as amodel red cell substitute, but was abandoned by the Army afterdemonstration of severe increases in pulmonary and systemic vascularresistance (Hess, J., V. Macdonald, A. Murray, V. Coppes, and C. Gomez,“Pulmonary and systemic hypertension after hemoglobin administratio,”Blood 78: 356A (1991)). A commercial version of this product was alsoabandoned after a disappointing Phase III clinical trial (Winslow, R. M.“αα-Crosslinked hemoglobin: Was failure predicted by preclinicaltesting?” Vox sang 79: 1-20 (2000).

The most commonly advanced explanation for the vasoconstriction producedby cell-free hemoglobin is that it readily binds the endothelium-derivedrelaxing factor, nitric oxide (NO). In fact, recombinant hemoglobinswith reduced affinity for NO have been produced which appear to be lesshypertensive in top-load rat experiments (Doherty, D. H., M. P. Doyle,S. R. Curry, R. J. Vali, T. J. Fattor, J. S. Olson, and D. D. Lemon,“Rate of reaction with nitric oxide determines the hypertensive effectof cell-free hemoglobin,” Nature Biotechnology 16: 672-676 (1998))(Lemon, D. D., D. H. Doherty, S. R. Curry, A. J. Mathews, M. P. Doyle,T. J. Fattor, and J. S. Olson, “Control of the nitric oxide-scavengingactivity of hemoglobin,” Art Cells, Blood Subs., and Immob. Biotech 24:378 (1996)). However, studies suggest that NO binding may not be theonly explanation for the vasoactivity of hemoglobin. It has been foundthat certain large hemoglobin molecules, such as those modified withpolyethylene glycol (PEG), were virtually free of the hypertensiveeffect, even though their NO binding rates were identical to those ofthe severely hypertensive ααHb (Rohlfs, R. J., E. Bruner, A. Chiu, A.Gonzales, M. L. Gonzales, D. Magde, M. D. Magde, K. D. Vandegriff, andR. M. Winslow, “Arterial blood pressure responses to cell-freehemoglobin solutions and the reaction with nitric oxide,” J Biol Chem273: 12128-12134 (1998)). Furthermore, it was found that PEG-hemoglobinwas extraordinarily effective in preventing the consequences ofhemorrhage when given as an exchange transfusion prior to hemorrhage(Winslow, R. M., A. Gonzales, M. Gonzales, M. Magde, M. McCarthy, R. J.Rohlfs, and K. D. Vandegriff, “Vascular resistance and the efficacy ofred cell substitutes,” J Appl Physiol 85: 993-1003 (1998)).

This protective effect correlated with the lack of hypertension,suggesting that vasoconstriction is responsible for the disappointingperformance of many of the hemoglobin-based products studied to date.Based on these observations, a hypothesis was developed to explainvasoconstriction, as an alternative, or possibly in addition to, theeffect of NO binding. Although not wishing to be bound by any particulartheory, it is believed that a substantial component of hemoglobin'svasoactive effect is a reflexive response to the diffusion of hemoglobinin the cell-free space. This hypothesis was tested in an in vitrocapillary system, and it was demonstrated that PEG-hemoglobin, which hasa reduced diffusion constant, transferred O₂ in a manner very similar tothat of native red blood cells (McCarthy, M. R., K. D. Vandegriff, andR. M. Winslow, “The role of facilitated diffusion in oxygen transport bycell-free hemoglobin: Implications for the design of hemoglobin-basedoxygen carriers,” Biophysical Chemistry 92: 103-117 (2001)). Oxygenaffinity would be expected to play a role in its facilitated diffusionby hemoglobin in the plasma space, since the change in saturation fromthe hemoglobin to the vessel wall is a determinant of the diffusiongradient of the hemoglobin itself.

Oxygen affinity of cell-free hemoglobin may play an additional role inthe regulation of vascular tone, since the release of O₂ to vessel wallsin the arterioles will trigger vasoconstriction (Lindbom, L., R. Tuma,and K. Arfors, “Influence of oxygen on perfusion capillary density andcapillary red cell velocity in rabbit skeletal muscle,” Microvasc Res19: 197-208 (1980)). In the hamster skinfold, the PO₂ in such vessels isin the range of 20-40 Torr, where the normal red cell oxygen equilibriumcurve is steepest (Intaglietta, M., P. Johnson, and R. Winslow,“Microvascular and tissue oxygen distribution,” Cardiovasc Res 32:632-643 (1996)). Thus from a theoretical point of view, it may beimportant for the P50 of cell-free hemoglobin to be lower than that ofred cells (i.e., higher O₂ affinity), in order to prevent release of O₂in arteriolar regulatory vessels.

Oxygen-Carrying Component

In preferred embodiments, the oxygen carrier (i.e., the oxygen-carryingcomponent) is a hemoglobin-based oxygen carrier, or HBOC. The hemoglobinmay be either native (unmodified); subsequently modified by a chemicalreaction such as intra- or inter-molecular cross-linking,polymerization, or the addition of chemical groups (e.g., polyalkyleneoxides, or other adducts); or it may be recombinantly engineered. Humanalpha- and beta-globin genes have both been cloned and sequenced.Liebhaber, et al., P.N.A.S. 77: 7054-7058 (1980); Marotta, et al., J.Biol. Chem. 353: 5040-5053 (1977) (beta-globin cDNA). In addition, manyrecombinantly produced modified hemoglobins have now been produced usingsite-directed mutagenesis, although these “mutant” hemoglobin varietieswere reported to have undesirably high oxygen affinities. See, e.g.,Nagai, et al., P.N.A.S., 82: 7252-7255 (1985).

The present invention is not limited by the source of the hemoglobin.For example, the hemoglobin may be derived from animals and humans.Preferred sources of hemoglobin for certain applications are humans,cows and pigs. In addition, hemoglobin may be produced by other methods,including chemical synthesis and recombinant techniques. The hemoglobincan be added to the blood product composition in free form, or it may beencapsulated in a vessicle, such as a synthetic particle, microballoonor liposome. The preferred oxygen-carrying components of the presentinvention should be stroma free and endotoxin free. Representativeexamples of oxygen-carrying components are disclosed in a number ofissued United States Patents, including U.S. Pat. No. 4,857,636 to Hsia;U.S. Pat. No. 4,600,531 to Walder, U.S. Pat. No. 4,061,736 to Morris etal.; U.S. Pat. No. 3,925,344 to Mazur; U.S. Pat. No. 4,529,719 to Tye;U.S. Pat. No. 4,473,496 to Scannon; 4,584,130 to Bocci et al.; U.S. Pat.No. 5,250,665 to Kluger et al.; U.S. Pat. No. 5,028,588 to Hoffman etal.; and U.S. Pat. No. 4,826,811 and U.S. Pat. No. 5,194,590 to Sehgalet al.

In addition to the aforementioned sources of hemoglobin, it has recentlybeen found that horse hemoglobin has certain advantages as the oxygencarrying component in the compositions of the present invention. Oneadvantage is that commercial quantities of horse blood are readilyavailable from which horse hemoglobin can be purified. Anotherunexpected advantage is that horse hemoglobin exhibits chemicalproperties that may enhance its usefulness in the blood substitutes ofthe present invention.

Previous reports have indicated that horse hemoglobin auto-oxidizes tomethemoglobin faster than human hemoglobin, which would make it lessdesirable as a blood substitute component. See, e.g., J. G. McLean andI. M. Lewis, Research in Vet. Sci., 19:259-262 (1975). In order tominimize auto-oxidation, McLean and Lewis used a reducing agent,glutathione, after red blood cell lysis. However, the hemoglobin that isused to prepare the compositions of the present invention, regardless ofwhether the source of hemoglobin is human or horse, do not require theuse of reducing agents to prevent auto-oxidation after red blood celllysis.

More recently, it has been reported that horse hemoglobin has an oxygenaffinity that is different from that of human hemoglobin. See, e.g., M.Mellegrini, et al., Eur. J. Biochem., 268: 3313-3320 (2001). Such adifference would discourage the selection of horse hemoglobin to prepareblood substitutes that mimic human hemoglobin. However, whenincorporated into the compositions of the present invention, nosignificant difference (less than 10%) in oxygen affinity between humanand horse hemoglobin-containing conjugates is observed.

Accordingly, contrary to these seemingly undesirable properties, in thecompositions of the present invention, horse hemoglobin is equivalent ifnot superior to human hemoglobin.

For use in the present invention, the HBOC has an oxygen affinity thatis greater than whole blood, and preferably twice that of whole blood,or alternatively, greater than that of stroma-free hemoglobin (SFH),when measured under the same conditions. In most instances, this meansthat the HBOC in the blood substitute will have a P50 less than 10, andmore preferably less than 7. In the free state, SFH has a P50 ofapproximately 15 torr, whereas the P50 for whole blood is approximately28 torr. It has previously been suggested that increasing oxygenaffinity, and thereby lowering the P50, may enhance delivery of oxygento tissues, although it was implied that a P50 lower than that of SFHwould not be acceptable. See Winslow, et al., in “Advances in BloodSubstitutes” (1997), Birkhauser, ed., Boston, Mass., at page 167, andU.S. Pat. No. 6,054,427. This suggestion contradicts the widely heldbelief that modified hemoglobins for use as blood substitutes shouldhave lower oxygen affinities, and should have P50s that approximate thatof whole blood. Hence, many researchers have used pyridoxyl phosphate toraise the P50 of SFH from 10 to approximately 20-22, since pyridoxylatedhemoglobin more readily releases oxygen when compared to SFH.

There are many different scientific approaches to manufacturing HBOCswith high oxygen affinity (i.e. those with P50s less than SFH). Forexample, studies have identified the amino acid residues that play arole in oxygen affinity, such as B-93 Cysteine, and thus site-directedmutagenesis can now be easily carried out to manipulate oxygen affinityto the desired level. See, e.g., U.S. Pat. No. 5,661,124. Many otherapproaches are discussed in U.S. Pat. No. 6,054,427.

Hemoglobin-Associated Toxicity

Hemoglobin is known to exhibit autooxidation when it reversibly changesfrom the ferrous (Fe²⁺) to the ferrie (Fe³⁺) or methemoglobin form. Whenthis happens, molecular oxygen dissociates from the oxyhemoglobin in theform of a superoxide anion (O₂—). This also results in destabilizationof the heme-globin complex and eventual denaturation of the globinchains. Both oxygen radical formation and protein denaturation arebelieved to play a role in vivo toxicity of HBOCs (Vandegriff, K. D.,Blood Substitutes, Physiological Basis of Efficacy, pages 105-130,Winslow et al., ed., Birkhauser, Boston, Mass. (1995).)

With most HBOCs, there is a negative correlation between oxygen affinityand hemoglobin oxidation, i.e., the higher the oxygen affinity, thelower the rate of autooxidation. However, the effects of differenthemoglobin modifications on oxygen affinity and the rate ofautooxidation are not always predictable. In addition, the optimalbalance between oxygen affinity and autooxidation rate is not wellunderstood.

The present invention relates, in part, to the unexpected finding thatthe PEG-Hb conjugates described herein exhibit very low rates ofautooxidation. When measured as a rate of oxidation, this value shouldbe as low as possible (i.e., 0.2% per hour of total hemoglobin, morepreferably 0.1% per hour of total hemoglobin, at room temperature for atleast 3 hours, and more preferably at least 10 hours. Thus, the HBOCs ofthe present invention remain stable during administration and/or storageat room temperature.

Modifications of the Oxygen-Carrying Component

In a preferred embodiment, the oxygen-carrying component is modifiedhemoglobin. A preferred modification to hemoglobin is“surface-modification,” i.e. covalent attachment of chemical groups tothe exposed amino acid side chains on the hemoglobin molecule.

Modification is carried out principally to increase the molecular sizeof the hemoglobin, most often by covalent attachment of polymericmoieities such as synthetic polymers, carbohydrates, proteins and thelike. Generally, synthetic polymers are preferred.

Suitable synthetic hydrophilic polymers include, inter alia,polyalkylene oxide, such as polyethylene oxide ((CH₂CH₂O)_(n)),polypropylene oxide ((CH(CH₃)CH₂O)_(n)) or a polyethylene/polypropyleneoxide copolymer ((CH₂CH₂O)_(n)—(CH(CH₃)CH₂O)_(n)). Other straight,branched chain and optionally substituted synthetic polymers that wouldbe suitable in the practice of the present invention are well known inthe medical field.

Most commonly, the chemical group attached to the hemoglobin ispolyethylene glycol (PEG), because of its pharmaceutical acceptabilityand commercial availability. PEGs are polymers of the general chemicalformula H(OCH₂CH₂)_(n)OH, where n is generally greater than or equal to4. PEG formulations are usually followed by a number that corresponds totheir average molecular weight. For example, PEG-200 has an averagemolecular weight of 200 and may have a molecular weight range of190-210. PEGs are commercially available in a number of different forms,and in many instances come preactivated and ready to conjugate toproteins.

An important aspect of preferred embodiments of the present invention isthat surface modification takes place when the hemoglobin is in theoxygenated or “R” state. This is easily accomplished by allowing thehemoglobin to equilibrate with the atmosphere (or, alternatively, activeoxygenation can be carried out) prior to conjugation. By performing theconjugation to oxygenated hemoglobin, the oxygen affinity of theresultant hemoglobin is enhanced. Such a step is generally regarded asbeing contraindicated, since many researchers describe deoxygenationprior to conjugation to diminish oxygen affinity. See, e.g., U.S. Pat.No. 5,234.903.

Although in many respects the performance of surface modifiedhemoglobins is independent of the linkage between the hemoglobin and themodifier (e.g. PEG), it is believed that more rigid linkers such asunsaturated aliphatic or aromatic C₁ to C₆ linker substituents mayenhance the manufacturing and/or characteristics of the conjugates whencompared to those that have more flexible and thus deformable modes ofattachment.

The number of PEGs to be added to the hemoglobin molecule may vary,depending on the size of the PEG. However, the molecular size of theresultant modified hemoglobin should be sufficiently large to avoidbeing cleared by the kidneys to achieve the desired half-life.Blumenstein, et al., determined that this size is achieved above 84,000molecular weight. (Blumenstein, et al., in “Blood Substitutes and PlasmaExpanders,” Alan R. Liss, editors, New York, N.Y., pages 205-212(1978).) Therein, the authors conjugated hemoglobin to dextran ofvarying molecular weight. They reported that a conjugate of hemoglobin(with a molecular weight of 64,000) and dextran (having a molecularweight of 20,000) “was cleared slowly from the circulation andnegligibly through the kidneys,” but increasing the molecular weightabove 84,000 did not alter the clearance curves. Accordingly, asdetermined by Blumenstein, et al., it is preferable that the HBOC have amolecular weight of at least 84,000.

In one embodiment of the present invention, the HBOC is a “MalPEG,”which stands for hemoglobin to which malemidyl-activated PEG has beenconjugated. Such MalPEG may be further referred to by the followingformula:Hb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—O—CH₃)_(m)  Formula I

where Hb refers to tetrameric hemoglobin, S is a surface thiol group, Yis the succinimido covalent link between Hb and Mal-PEG, R is an alkyl,amide, carbamate or phenyl group (depending on the source of rawmaterial and the method of chemical synthesis), [O—CH₂—CH₂]_(n) are theoxyethylene units making up the backbone of the PEG polymer, where ndefines the length of the polymer (e.g., MW=5000), and O—CH₃ is theterminal methoxy group.

Crystalloid Component

In one embodiment of the present invention, the blood substitute mayalso comprise a crystalloid. The crystalloid component can be anycrystalloid which, in the form of the blood substitute composition, ispreferably capable of achieving an osmolarity greater than 800 mOsm/l,i.e. it makes the blood substitute “hypertonic”. Examples of suitablecrystalloids and their concentrations in the blood substitute include,e.g., 3% NaCl, 7% NaCl, 7.5% NaCl, and 7.5% NaCl in 6% dextran. Morepreferably, the blood substitute has an osmolarity of between 800 and2400 mOsm/l. The use of recombinantly produced hemoglobins in solutionswith an osmolality between 300-800 mOsm/l that further comprise acolloid (i.e. a molecule less diffusible than dextrose) have beenpreviously reported. See, e.g., U.S. Pat. No. 5,661,124. However, thispatent teaches away from producing blood substitutes with osmolalitiesabove 800, and suggests that the hemoglobin concentration should bebetween 6-12 g/dl. In contrast, the oxygen carrying efficiency ofcompositions of the present invention permit lower concentrations ofhemoglobin to be used, such as greater than 6 g/dl or even greater than4 g/dl. When the blood substitute further comprises a crystalloid and ishypertonic, the compositions of present invention may provide improvedfunctionality for rapid recovery of hemodynamic parameters over otherblood substitute compositions, which include a colloid component. Smallvolume highly hypertonic crystalloid infusion (e.g., 1-10 ml/kg)provides significant benefits in the rapid and sustained recovery ofacceptable hemodynamic parameters in controlled hemorrhage. (See, e.g.,Przybelski, R. J., E. K. Daily, and M. L. Birnbaum, “The pressor effectof hemoglobin—good or bad?” In Winslow, R. M., K. D. Vandegriff, and M.Intaglietta, eds. Advances in Blood Substitutes. IndustrialOpportunities and Medical Challenges. Boston, Birkhäuser (1997), 71-85).Hypertonic crystalloid solutions alone, however, do not adequatelyrestore cerebral oxygen transport. See D. Prough, et al., “Effects ofhypertonic saline versus Ringer's solution on cerebral oxygen transportduring resuscitation from hemorrhagic shock,” J. Neurosurg. 64:627-32(1986).

Formulation

The blood substitutes of the present invention are formulated by mixingthe oxygen carrier and other optional excipients with a suitablediluent. Although the concentration of the oxygen carrier in the diluentmay vary according to the application, and in particular based on theexpected post-administration dilution, in preferred embodiments, becauseof the other features of the compositions of the present invention thatprovide for enhanced oxygen delivery and therapeutic effects, it isusually unnecessary for the concentration to be above 6 g/dl, and ismore preferably between 0.1 to 4 g/dl.

Suitable diluents (i.e., one which is pharmaceutically acceptable forintravenous injection) include, intra alia, proteins, glycoproteins,polysaccharides, and other colloids. It is not intended that theseembodiments be limited to any particular diluent. Thus, it is intendedthat the diluent encompass aqueous cell-free solutions of albumin, othercolloids, or other non-oxygen carrying components, and the aqueoussolution has a viscosity of at least 2.5 cP. In some preferredembodiments, the viscosity of the aqueous solution is between 2.5 and 4cP. It is contemplated that the present invention also encompassessolutions with a viscosity of 6 cP or greater.

Applications

A. Clinical Applications

It is contemplated that the present invention and its embodiments willbe useful in applications where a rapid restoration of O₂ levels or anincreased O₂ level or a replacement of O₂ levels is clinicallyindicated. See, e.g., U.S. Pat. No. 6,054,427. The numerous settings inwhich the methods and compositions of the present invention find useinclude the following:

Trauma. An acute loss of whole blood can result in a fluid shift fromthe interstitial and intracellular spaces to replace the lost volume ofblood while shunting of blood away from the low priority organsincluding the skin and gut. Shunting of blood away from organs reducesand sometimes eliminates O₂ levels in these organs and results inprogressive tissue death. Rapid restoration of O₂ levels is contemplatedas perhaps resulting in a significantly better salvage of tissues inpatients suffering such acute blood loss.

Ischemia. In ischemia, a particular organ (or organs) are “starved” foroxygen. Small sections of the organ, known as infarcts, begin to die asa result of the lack of O₂. Rapid restoration of O₂ levels is criticalis stemming infarct formation in critical tissues. Conditions resultingin ischemia include heart attack, stroke, or cerbrovascular trauma.

Hemodilution: In this clinical application, a blood substitute isrequired to replace blood that is removed pre-operatively. It iscontemplated that the patient blood removal occurs to prevent arequirement for allogeneic transfusions post-operatively. In thisapplication, the blood substitute is administered to replace (orsubstitute for) the O₂ levels of the removed autologous blood. Thispermits the use of the removed autologous blood for necessarytransfusions during and after surgery. One such surgery requiringpre-operative blood removal would be a cardiopulmonary bypass procedure.

Septic Shock. In overwhelming sepsis, some patients may becomehypertensive in spite of massive fluid therapy and treatment withvasoconstrictor agents. In this instance, the overproduction of nitricoxide (NO) results in the lowered blood pressure. Therefore hemoglobinis close to an ideal agent for treatment of these patients becausehemoglobin binds NO with an avidity that parallels O₂.

Cancer. Delivery of O₂ to the hypoxic inner core of a tumor massincreases its sensitivity to radiotherapy and chemotherapy. Because themicrovasculature of a tumor is unlike that of other tissues,sensitization through increasing O₂ levels requires O₂ be unloadedwithin the hypoxic core. In other words, the P50 should be very low toprevent early unloading of the O₂, increasing the O₂ levels, to insureoptimal sensitization of the tumor to subsequent radiation andchemotherapy treatments.

Chronic anemia. In these patients, replacement of lost or metabolizedhemoglobin is compromised or completely absent. It is contemplated thatthe blood substitute must effectively replace or increase the reduced O₂levels in the patient.

Sickle cell anemia. In sickle cell anemia, the patient is debilitated bya loss of O₂ levels that occurs during the sickling process as well as avery high red blood cell turnover rate. The sickling process is afunction of PO₂ where the lower the PO₂, the greater the sickling rate.It is contemplated that the ideal blood substitute would restore patientO₂ levels to within a normal range during a sickling crisis.

Cardioplegia. In certain cardiac surgical procedures, the heart isstopped by appropriate electrocyte solutions and reducing patienttemperature. Reduction of the temperature will significantly reduce theP50, possibly preventing unloading of O₂ under any ordinaryphysiological conditions. Replacement of O₂ levels is contemplated aspotentially reducing tissue damage and death during such procedures.

Hypoxia. Soldiers, altitude dwellers, and world-class athletes underextreme conditions may suffer reduced O₂ levels because extraction of O₂from air in the lung is limited. The limited O₂ extraction furtherlimits O₂ transport. It is contemplated that a blood substitute couldreplace or increase the O₂ levels in such individuals.

Organ Perfusion. During the time an organ is maintained ex vivo,maintaining O₂ content is essential to preserving structural andcellular integrity and minimizing infarct formation. It is contemplatedthat a blood substitute would sustain the O₂ requirements for such anorgan.

Cell Culture. This requirement is virtually identical to that of organperfusion, except that the rate of O₂ consumption may be higher.

Hematopoiesis. It is contemplated that the blood substitute serves as asource for heme and iron for use in the synthesis of new hemoglobinduring hematopoiesis.

B. Veterinary Applications

The present invention can also be used in non-humans. The methods andcompositions of the present invention may be used with domestic animalssuch as livestock and companion animals (e.g, dogs, cats, horses, birds,reptiles), as well as other animals in aquaria, zoos, oceanaria, andother facilities that house animals. It is contemplated that the presentinvention finds utility in the emergency treatment of domestic and wildanimals suffering a loss of blood due to injury, hemolytic anemias, etc.For example, it is contemplated that embodiments of the presentinvention are useful in conditions such as equine infectious anemia,feline infectious anemia, hemolytic anemia due to chemicals and otherphysical agents, bacterial infection, Factor IV fragmentation,hypersplenation and splenomegaly, hemorrhagic syndrome in poultry,hypoplastic anemia, aplastic anemia, idiopathic immune hemolyticconditions, iron deficiency, isoimmune hemolytic anemia,microangiopathic hemolytic, parasitism, etc. In particular, the presentinvention finds use in areas where blood donors for animals of rareand/or exotic species are difficult to find.

EXAMPLES Example 1 Production of Stroma-Free Hemoglobin

Step-1—Procurement of Outdated Red Blood Cells

Outdated packed red blood cells are procured from a commercial source,such as the San Diego Blood Bank or the American Red Cross. Preferably,outdated material is received not more than 45 days from the time ofcollection. Packed RBCs (pRBCs) are stored at 4±2° C. until furtherprocessed (1-7 days). All units are screened for viral infection andsubjected to nucleic acid testing prior to use.

Step-2—Pooling of Outdated Blood

Packed red blood cells are pooled into a sterile vessel in a cleanfacility. Packed red blood cell volume is noted, and hemoglobinconcentration is determined using a commercially available co-oximeteror other art-recognized method.

Step-3—Leukodepletion

Leukodepletion (i.e. removal of white blood cells) is carried out usingmembrane filtration. Initial and final leukocyte counts are made tomonitor the efficiency of this process.

Step4—Cell Separation and Cell Wash

Red blood cells are washed with six volumes of 0.9% sodium chloride. Theprocess is carried out at 4±2° C. The cell wash is analyzed to verifyremoval of plasma components by a spectrophotometric assay for albumin.

Step-5—Red Blood Cell Lysis and Removal of Cell Debris

Washed red blood cells are lysed at least 4 hours or overnight at 4±2°C. with stirring using 6 volumes of water. Lysate is processed in thecold to purify hemoglobin. This is achieved by processing the lysatethrough a 0.16-μm membrane. Purified hemoglobin is collected in asterile depyrogenated vessel. All steps in this process are carried outat 4±2° C.

Step-6—Viral Removal

Viral removal is performed by ultrafiltration at 4±2° C.

Step-7—Concentration and Solvent Exchange

Hemoglobin purified from lysate and ultrafiltration is exchanged intoRinger's lactate (RL) or phosphate-buffered saline (PBS, pH 7.4) using a10-kD membrane. The hemoglobin is then concentrated using the samemembrane to a final concentration of 1.1-1.5 mM (in tetramer). Ten to 12volumes of RL or PBS are used for solvent exchange. This process iscarried out at 4±2° C. The pH of the solution prepared in RL is adjustedto 7.0-7.6.

Step-8—Sterile Filtration

Hemoglobin in PBS or Ringer's lactate(RL) is sterile-filtered through a0.45- or 0.2-μm disposable filter capsule and stored at 4±2° C. beforethe chemical modification reaction is performed.

Other methods for purifying hemoglobin are well known in the art. Inaddition, the use of a reducing agent (e.g., glutathione or anotherthiol-containing reducing agent) to prevent auto-oxidation after celllysis is usually unnecessary.

Example 2 Modification of Stroma Free Hemoglobin

Step-1—Thiolation

Thiolation is carried out using 10-fold molar excess iminothiolane overhemoglobin for 4 hours at 4±2° C. with continuous stirring.

Reaction conditions:

1 mM hemoglobin (tetramer) in RL (pH 7.0-7.5) or PBS (pH 7.4)

10 mM iminothiolane in RL (pH 7.0-7.5) or PBS (pH 7.4)

The ratio of 1:10 SFH:iminothiolane and reaction timing were optimizedto maximize the number of PEGylated thiol groups and to minimize productheterogeneity.

Step 2—PEGylation of Thiolated Hemoglobin

Thiolated hemoglobin is PEGylated using a 20-fold molar excess ofMal-PEG (with an alkyl or phenyl linker) based on starting tetramerichemoglobin concentration. The hemoglobin is first allowed to equilibratewith the atmosphere to oxygenate the hemoglobin. The reaction takesplace for 2 hours at 4±2° C. with continuous stirring.

Reaction conditions:

1 mM thiolated hemoglobin in RL or PBS (pH 7.4)

20 mM Mal-PEG in RL or PBS (pH 7.4)

Step 3—Removal of Unreacted Reagents

PEGylated-Hb is processed through a 70-kD membrane to remove excessunreacted reagents or hemoglobin. A 20-volume filtration is carried outto ensure removal of unreacted reagents, which is monitored bysize-exclusion chromatography at 540 nm and 280 nm. The proteinconcentration is diluted to 4 g/dl. The pH is adjusted to 7.3±0.3 using1 N NaOH.

Step-3—Sterile Filtration

The final MalPEG-Hb product is sterile-filtered using a 0.2-μm steriledisposable capsule and collected into a sterile depyrogenated vessel at4±2° C.

Step-4—Formulation of MalPEG-Hb

PEGylated Hb is diluted to 4 g/dl RL, pH adjusted to 7.4±0.2

Step-5—Sterile Fill

The final blood substitute composition is sterile-filtered (0.2 μm) andaliquoted by weight into sterile glass vials and closed with sterilerubber stoppers with crimped seals in a laminar flow hood and stored at−80° C. until use.

Example 3 Physiochemical Analysis of MalPEG-Hb

Methodology for Physiochemical Analysis

Homogeneity and molecular size of the MalPEG-Hb blood substitute arecharacterized by Liquid Chromatography (LC). Analytical LC is used toevaluate homogeneity of the PEGylated hemoglobin and extent of removalof unreacted Mal-PEG. Absorbance at 540 nm is used to evaluatehemoglobin and resolves PEGylated hemoglobin from unreacted hemoglobinby peak position. Absorbance at 280 nm is used to resolve PEGylatedhemoglobin from free Mal-PEG, which absorbs in the ultraviolet (UV)spectrum due to the ring structures in MalPEG.

Optical spectra are collected using a rapid scanning diode arrayspectrophotometer (Milton Roy 2000 or Hewlett Packard Model 8453) in theSoret and visible regions for analysis of hemoglobin concentration andpercent methemoglobin by multicomponent analysis (Vandegriff, K. D., andR. E., Shrager. Evaluation of oxygen equilibrium binding to hemoglobinby rapid-scanning spcetrophotometry and singular value decomposition.Meth. Enzymol. 232: 460-485 (1994).

MalPEG-Hb concentration and percentage methemoglobin are determinedusing a co-oximeter. Viscosity is determined using a Rheometer. ColloidOsmotic Pressure is determined using a colloid osmometer. Oxygen bindingparameters are determined from oxygen equilibrium curves.

The preferred specifications for the blood substitute composition arepresented in Table 1 below:

TABLE 1 Test Specification Hemoglobin concentration (g/dl) 4.2 ± 0.2Methemoglobin (%) <10 pH 7.4 ± 0.4 Conductivity (mS/cm) 12 ± 4 Endotoxin (EU/mL) <0.5 FPLC retention time (min) 43 ± 3  FPLC peak widthat half height (min) 6 ± 2 Viscosity (cPs) 2.5 ± 1.0 COP (mmHg) 50 ± 20P50 (Torr) 6 ± 2 Hill number (at P50) 1.2 ± 0.5 Sterility PassNumber of PEGylated Sites on MalPEG-Hb

For surface modification, the number “m” in Formula I is the parameterthat defines the number of PEG polymers attached to the surface ofhemoglobin.Hb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—CH₃)_(m)  Formula I

To determine this number, a dithiopyridine colorimetric assay (Ampulski,R., V. Ayers, and S. Morell. Determination of the reactive sulfhydrylgroups in heme proteins with 4,4′-dipyridinesdisulde. Biocheim. Biophys.Acta 163-169, 1969) is used to measure the number of available thiolgroups on the surface of the Hb tetramer before and after thiolation andthen again after Hb PEGylation. Human hemoglobin contains 2 intrinsicreactive thiol groups at the β93Cys residues, which is confirmed by thedithiopyridine reaction. After thiolation of SFH at a ratio of 1:10SFH:iminothiolane, the number of reactive thiol groups increases from 2to 6 thiols based on the dithiopyridine reaction. After the PEGylationreaction, the number of reactive thiol groups is decreased to 1.3. Thisindicates that there are 4-5 PEGylated sites on MalPEG-Hb.

Size-exclusion Chromatography Analysis of MalPEG-Hb Versus SFH

FPLC is performed for analysis of the final MalPEG-Hb product. Typicalchromatograms are displayed in FIG. 1 for MalPEG-Hb compared tounmodified SFH. The retention time for SFH is approximately 57 min. Theretention time for MalPEG-Hb is approximately 44 min.

Physical and Chemical Characteristics of MalPEG-Hb

The physical properties of MalPEG-Hb compared to blood and unmodifiedhuman hemoglobin (SFH) are shown below in Table 2.

TABLE 2 Blood SFH MalPEG-Hb P50 (Torr) 28 15 5 N50 (Hill number) 2.9 2.91.2 Bohr effect (ΔLog — −0.46 −0.20 P50/ΔpH) Viscosity (cPs)¹ 4.0 0.92.5 COP (mm Hg)¹ 27 16 50 MW (kD)² N/A 65 90 Molecular Radius (nm) 40003.2² 9 ¹Determined at 15 g/dl for whole blood and approximately 4 g/dlfor hemoglobin solutions ²Determined by COP measurements and FPLCOxygen Affinity

Hemoglobin-oxygen equilibrium binding curves were measured as describedpreviously (Vandegriff, K. D., R. K. Rohlfs, M. D. Magde, and R M.Winslow. Hemoglobinoxygen equilibrium cures measured during enzymaticoxygen consumption. Anal. Biochem. 256: 107-116, 1998). MalPEG-Hbexhibits a high oxygen affinity (P50=5 mm Hg) and low cooperativity(n50=1.0-1.4). FIG. 2 shows representative curves comparing stroma-freehemoglobin (SFH) and MalPEG-Hb solutions.

Viscosity

This solution property of MalPEG-Hb is due to the strong interactionbetween polyethylene glycol chains and solvent water molecules. This isbelieved to be an important attribute for a blood substitute for tworeasons: 1) higher viscosity decreases the diffusion constant of boththe PEG-Hb molecule and gaseous ligand molecules diffusing through thesolvent, and 2) higher viscosity increases the shear stress of thesolution flowing against the endothelial wall, eliciting the release ofvasodilators to counteract vasoconstriction. As shown in Table 2, theviscosity of the MalPEG-Hb solution is 2.5 cPs.

Colloidal Osmotic Pressure (COP)

The COP of hemoglobin solutions containing unmodified, intra- andintermolecularly cross-linked, or PEG-surface-conjugated hemoglobin havebeen measured to determine their macromolecular solution properties(Vandegriff, K. D., R. J. Rohlfs, and R. M. Wislow. Colloid osmoticeffects of hemoglobin-based oxygen carriers. In Winslow, R. M., K. D.Vandegriff and M. Intaglia, eds, Advances in Blood SubstitutesIndustrial Opportunities and Medical Challenges. Boston, Birkhauser, pp.207-232 (1997). Tetrameric hemoglobins show nearly ideal solutionbehavior; whereas hemoglobins conjugated to PEG have significantlyhigher colloid osmotic activity and exhibit solution non-ideality(Vandegriff, K. D., M. Mcarthy, R. J. Rohls and R. M. Winslow. Colloidosmotic properties of modified hemoglobins: chemically cross-linkedversus polyethylene glycol surface-conjugated. Biophys. Chem. 69:23-30(1997). As shown in Table 2, the COP of the MalPEG-Hb solution is50.

Stability

The stability of hemoglobin solutions containing PEG-surface-conjugatedhemoglobin have been determined by examining the rate of autoxidation.At room temperature, the autoxidation of MalPEG-Hb increased fromapproximately 5% MetHb to 5.5% MetHb in 10 hours as shown in FIG. 5. Theautoxidation rate for MalPEG-Hb was 0.05% per hour.

Example 4 Comparison of Modified Hemoglobins with Different P50s

The role of oxygen affinity in the efficacy of cell-free hemoglobinusing hemoglobin modified by conjugation to polyoxyethylene (POE) is ofparticular interest in studying the efficacy of such materials as bloodsubstitutes. This modification, first described by Iwashita andcoworkers (Ajisaka, K. and Y. Iwashita, “Modification of humanhemoglobin with polyethylene glycol: A new candidate for bloodsubstitute. BBRC 97: 1076-1081 (1980)) (Iwasaki, K., K. Ajisaka, and Y.Iwashita, “Modification of human hemoglobin with polyoxyethylene glycol:A new candidate for blood substitutes,” Biochem Biophys Res Comm 97:1076-1981 (1980)), retains a hypertensive effect, and has been found tobe useful in the treatment of septic shock. As part of the preparationof this product, hemoglobin was reacted with pyridoxal-5-phosphate (PLP)to raise its P50, close to the value for human blood. Thus it waspossible to prepare two solutions of POE-modified hemoglobin, one withand one without prior modification with PLP. These solutions areidentical in every way except for their P50, and were tested for theirability to support physiological function in rats during a severe (60%of blood volume) hemorrhage.

Materials and Methods:

Blood Substitutes

The modified hemoglobin solutions, with or without PLP modification toform “PHP” were prepared as described above in Example 1.

Animals

Male Sprague-Dawley rats were used for this study. Systolic anddiastolic pressures were monitored during the study; the maximum andminimum pressures, respectively, and the mean arterial pressure (MAP)was diastolic +1/3 (systolic-diastolic) pressure. The dP/dt wascalculated from the maximum positive slope for each pressure cycle. Meanvalues of heart rate, systolic, diastolic, mean arterial pressures,pulse pressure and dP/dt were averaged for each minute of data.

Blood Gases, Hematologic, and Lactate Measurements

Arterial pH, PCO₂, and PO₂ were measured in a blood gas analyzer using100 μl heparinized samples of blood. Lactic acid was measured in arteryblood using a Lactate Analyzer. Total CO₂, standard bicarbonate (HCO₃⁻), and base excess (BE) were calculated from PCO₂, pH and hemoglobinconcentration using algorithms described previously (Winslow, R., “Amodel for red cell O_(2 uptake) ,”. Int J Clin Monit Comput 2: 81-93(1985)). Total hemoglobin and plasma hemoglobin were each measured usingcommercially available equipment. Hematocrit was measured usingapproximately 50 μl samples of arterial blood by microcentrifugation.

Exchange Transfusion

Exchange-transfusion was carried out at a rate of approximately 0.5ml/min to a total volume of solution that equaled 50% of estimated bloodvolume. Blood volume was assumed to be 65 ml/kg. The peristaltic pumpwas operated so that blood was removed at exactly the same rate as testmaterial was infused. Test solutions were warmed to 37° C. in a waterbath prior to infusion and kept warm during infusion.

Hemorrhage

The hemorrhage protocol we used is based on the model of Hannon and Wade(Hannon, J., C. Wade, C. Bossone, M. Hunt, R. Coppes, and J. Loveday,“Blood gas and acid-base status of conscious pigs subjected tofixed-volume hemorrhage and resuscitated with hypertonic sali dextran,”Circ Shock 32: 19-29 (1990)). Hemorrhage was begun approximately 3minutes after completion of the exchange transfusion by pumping outarterial blood from the femoral artery at a rate of 0.5 ml/min to remove60% of the blood volume by the end of 60 minutes. Blood samples (0.3 ml)were taken every 10 minutes for hematologic and blood gas analysis.

Statistical and Survival Analysis

For the survival analyses, animals were observed for a minimum of 120minutes after the start of the hemorrhage. The data were grouped into10-minute intervals, and for each interval the cumulative proportionalive and its standard error were calculated.

Results:

Solution Properties

The solutions used are described below in Table 3. The total hemoglobinconcentration, viscosity and colloid osmotic pressure (COP) arewell-matched. The P50 of the PHP (19.7 Torr) was higher than that of thePOE (12.2 Torr). The degree of cooperativity (Hill's parameter, n) wereequivalent for the two solutions.

The FPLC patterns for the two solutions are given in FIG. 3. While thereis a small peak in each that corresponds to unmodified hemoglobin, thebulk of the hemoglobin appears in a heterogeneous set of peaks thatelute significantly earlier than the unmodified hemoglobin (SFH). Thepatterns for the two PEG-modified hemoglobins are qualitatively similar.

TABLE 3 Table 1. Properties of the test solutions PHP POE Hb, g/dl 8.08.3 Viscosity (cP) 2.8 2.8 COP, mm Hg 62.7 56.5 *a₁ (×10⁻¹) 1.368 2.228*a₂ (×10⁻³) 9.680 21.070 *a₃ (×10⁻⁵) 0.752 46.500 *a₄ (×10⁻⁵) 1.5378.766 P50 19.7 12.2 n50 1.48 1.49 *Oxygen Affinity Measured at 37° C.,pH 7.4

The oxygen affinity of the POE was significantly higher than that of thePHP (FIG. 4). Neither product, however, displays significantcooperativity.

Animal Experiments

All experiments are summarized in Table 4. A larger number of animalsreceived PHP (18) than POE (11), and the average weight of the PHPanimals was significantly greater than that of the POE group (P<0.001).However, this difference in weight was accounted for in calculating thedegree of hemorrhage, assuming a total blood volume of 65 ml/kg.Therefore the consequent volumes of exchange transfusion and hemorrhagewere different as well. Nevertheless, the mean time to death wassignificantly shorter for the PHP animals (93 minutes) compared to thePOE animals (116 minutes). This difference was statistically significant(P<0.02). If animals survived the observation period, 120 minutes afterstart of hemorrhage, they were considered “censored” for the purpose ofthe Kaplan-Meier survival analysis (FIG. 5).

TABLE 4 *Blood Hemorrhage Hemorrhage Time to WT Volume Volume VolumeDeath (g) (ml) (ml) (%) (minutes) PHP n 18 18 18 18 18 PHP 291 18.8911.10 58.79 93 sd 24 1.59 0.90 0.35 29 POE n 11 11 11 11 11 POE 33421.74 12.78 58.77 116 sd 41 2.65 1.60 0.39 12 P 0.001 0.001 0.001 0.9150.020 *Based on 65 ml/kg total blood volumeHematology and Acid-base Regulation

The baseline, post-ET and post-hemorrhage (60 minute) measurements areshown in Table 5. The mean hematocrit was slightly higher in the POEcompared to PHP animals, but after exchange transfusion the values wereidentical in the two groups. At the end of the hemorrhage period, themean hematocrit in the POE animals was again slightly higher than in thePHP animals. Similar minor differences are found in the total hemoglobinvalues, with POE animals being slightly higher at all sampling points.Plasma hemoglobin was not different in the two groups, but wassignificantly higher in the POE animals after the exchange period.

Arterial lactic acid concentration was significantly higher in the POEcompared to PHP animals at all stages of the study. The base excessvalues were not significantly different between the two groups, althoughthere is a suggestion that the values are lower in the PHP compared toPOE groups. Furthermore, the difference between baseline values for thePHP animals (10.24 mEq/l) is higher than for the POE animals (7.01mEq/l).

TABLE 5 n PHP sem n POE sem P HCT Baseline 17 39.80 0.57 10 43.15 0.230.0032 Post ET 17 17.89 0.43 10 19.25 0.62 0.3568 60 minutes 15 13.290.47 10 15.85 0.14 0.0015 HB Baseline 17 13.59 0.22 10 15.08 0.17 0.0057Post ET 17 8.70 0.18 10 9.74 0.07 0.0007 60 minutes 15 6.34 0.21 10 7.380.06 0.0016 PLHB Baseline 16 0.00 0.00 10 0.00 0.00 Post ET 16 2.86 0.0710 2.99 0.09 0.6785 60 minutes 15 1.93 0.07 10 2.47 0.02 0.0001 LACTBaseline 9 0.70 0.06 7 2.68 0.16 0.0001 Post ET 9 1.59 0.10 7 4.21 0.240.0004 60 minutes 9 10.62 0.89 7 17.27 1.18 0.0360 BE Baseline 17 6.221.28 9 5.41 0.08 0.6530 Post ET 17 6.20 1.41 5 5.60 0.13 0.8101 60minutes 15 −4.02 1.60 6 −1.60 0.53 0.4678Mean Arterial Pressure during Exchange Transfusion

The mean arterial pressure during exchange transfusion is shown in FIG.6. Baseline mean arterial pressures are indistinguishable for the twogroups. However, the blood pressure response to infusion of thePEG-hemoglobins is significantly different between the two groups. Theinitial rise in MAP is greater in the PHP compared to the POE animals,and it is sustained for the duration of the infusion period. Incontrast, the MAP in the POE animals returns to baseline by the end ofthe infusion.

At the start of hemorrhage, the fall in MAP is immediate in the PHPanimals and delayed in the POE group. Furthermore, the MAP is sustainedat or near baseline values for the entire hemorrhage and beyond for thePOE animals, while the MAP never returns to baseline values in the PHPanimals. Especially in the PHP animals, the scatter in the data, asindicated by increasing standard errors, increases with time as animalsdrop out of the PHP group.

Discussion:

In this study, we studied 2 closely matched modified hemoglobinsolutions (“blood substitutes”) with respect to their ability to protectrats from a severe (60% of blood volume) hemorrhage. In order to testthis ability, animals first received a 50% (of blood volume) exchangetransfusion with one of the two test solutions. The solutions themselvesdiffered only in their oxygen affinity, and were matched very closely inFPLC pattern, oncotic pressure, viscosity and concentration. Otherstudies attempting to show the effects of specific variables onphysiological outcomes have not been able to compare solutions as wellmatched. See. e.g., Sakai, H., Hara, H., Tsai, A. G., Tsuchida, E.,Johnson, P. C., and Intaglietta, M., “Changes in resistance vesselsduring hemorrhagic shock and resuscitation in conscious hamster model,”Am J. Physiol 276(45), H563-H571. (1999), Sakai, H., H. Hara, M. Yuasa,A. Tsai, S. Takeoka, E. Tsuchida, and M. Intaglietta, “Moleculardimensions of Hb-based O₂ carriers determine constriction of resistancearteries and hypertension,” Am J Physiol 279: H908-H915, (2000). Theseexperiments represent the first instance in which such closely matchedsolutions could be compared with only one variable, P50, beingsignificantly different.

The group of animals that received POE had slightly, but significantly,higher hematocrits, total hemoglobins and plasma hemoglobin levels.However, it is very unlikely that these differences can explain theoutcome or interpretation of the experiments. Clearly, the two solutionsaffect blood pressure in different ways, as shown in FIG. 4. At the timeof infusion, the effect on blood pressure must be a function of theproperties of the infused solution, not the recipient animals. The bloodpressure response is greater and sustained in the PHP compared to POEanimals.

Survival of the animals is clearly not linked to the pressor effect ofthe hemoglobin solutions, as has been suggested by some investigators inthe past, because survival (and a suggestion of less base deficit) isbetter in the POE compared to PHP animals, in which the blood pressureeffect is less and only transient. See Przybelski, R. J., E. K. Daily,and M. L. Birnbaum, “The pressor effect of hemoglobin—good or bad?” InWinslow, R. M., K. D. Vandegriff, and M. Intaglietta, eds. Advances inBlood Substitutes. Industrial Opportunities and Medical Challenges.Boston, Birkhäuser (1997), 71-85.

Taken together, these results support the hypothesis that a lower P50 isbeneficial for the use of cell-free hemoglobin as an oxygen carrier. Thehypothesis is based on 2 concepts. First, that the diffusion gradientfor cell-free hemoglobin is a function of the oxyhemoglobin gradientbetween the source of oxygen, the red cell, and the vessel wall. Thisgradient, in turn is dependent on the shape and position of the oxygenequilibrium curve (McCarthy, M. R., K. D. Vandegriff, and R. M. Winslow,“The role of facilitated diffusion in oxygen transport by cell-freehemoglobin: Implications for the design of hemoglobin-based oxygencarriers,” Biophysical Chemistry 92: 103-117 (2001)). Second, thisconsideration leads to the second conceptual basis of our hypothesis,that high oxygen affinity (low P50) effectively “hides” O₂ from thecirculation until the carrier hemoglobin molecule arrives at regions ofthe circulation in which the PO₂ is very low, such as in ischemic orhypoxic tissue.

Example 5 Stability of MalPEG-Hb

The purpose of this study was to determine the stability of MalPEG-Hbduring a simulation of the storage and handling conditions of thesamples for Phase I clinical trials. The stability during three stagesof handling were assessed. Stage I represented the transfer from frozenstorage at the production facility to temperature conditions duringshipping to the clinical site (frozen storage study). Stage IIrepresented the thawing of the MalPEG-Hb for 24 hours to +4° C. andsubsequent storage at +4° C. for five days (refrigerated study). StageIII represented the thawing of the MalPEG-Hb for 24 hours to +4° C. andsubsequent storage of MalPEG-Hb at room temperature for several daysprior to patient administration (room temperature study).

Experimental Methods

Stability was defined by the rate of oxidation of the MalPEG-Hb testmaterial. The percentage of methemoglobin in the sample was measuredusing co-oximetry (IL Co-oximetry 682). Measurements were made induplicate at each time point according to the protocol.

Temperatures were monitored by thermometer or temperature chartrecorders. The frozen storage study was conducted over a temperaturerange of −21.0±3.0° C. The refrigerated study was conducted over atemperature range of +4.0±0.2° C. The room temperature study wasconducted over a temperature range of +21.0±1.0° C.

Temperature, total hemoglobin, and percent methemoglobin were recordedat each of the indicated time points. In the frozen and refrigeratedstudies, measurements were taken at time zero (completely thawed), onehour later, and then every 24 hours for five days. In the roomtemperature study, measurements were taken at time zero (completelythawed) and subsequently every one hour for ten hours.

Results

MalPEG-Hb showed no change in percent methemoglobin during 6 day storageat −20° C. as shown in FIG. 8. Similarly, MalPEG-Hb showed no change inpercent methemoglobin during five day storage at +4° C. as shown in FIG.9. During storage at room temperature, MalPEG-Hb showed less than 1percent increase in methemoglobin over a ten hour period as shown inFIG. 10.

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the compositions, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes for carrying outthe invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each suchpublication, patent or patent application were specifically andindividually indicated to be incorporated herein by reference.

1. A method of making a blood substitute product comprising the stepsof: a) preparing an oxygenated hemoglobin solution; b) thiolating theoxygenated hemoglobin to form thiolated oxygenated hemoglobin; c)reacting the thiolated oxygenated hemoglobin with maleimidylpolyethylene glycol to form polyethylene glycol modified hemoglobin;wherein the polyethylene glycol modified hemoglobin has a P50 less thantwo thirds that of native stroma-free hemoglobin from the same animalsource when measured under the same conditions.
 2. The method of claim1, wherein the polyethylene glycol modified hemoglobin has a P50 lessthan 10 torr.
 3. The method of claim 1, wherein the polyethylene glycolmodified hemoglobin has a P50 of 6+/−2 torr.
 4. The method of claim 1,wherein the concentration of hemoglobin is between 0.1 to 4.0 g/dl. 5.The method of claim 1, wherein the viscosity is 2.5+/−1.0 cPs.
 6. Themethod of claim 1, wherein the polyethylene glycol modified hemoglobinhas a P50 less than 6 torr.
 7. The method of claim 1, wherein thepolyethylene glycol attached to the polyethylene glycol modifiedhemoglobin is attached to surface thiol groups on the hemoglobin.
 8. Themethod of claim 7, wherein at least some of the surface thiol groups arechemically added to the hemoglobin prior to attachment of thepolyethylene glycol.